Using a tumor virus, we are defining the events that occur during the initiation of DMAreplication. Initiation of DNA replication is a complicated process that is known to take place in many stages; these include the recognition of the viral origin by the virally encoded initiator protein, subsequent oligomerization events that are coupled to structural distortions of the origin, transition of the initiator into a 3'->5' helicase and recruitment of cellular factors for subsequent steps in initiation. Recent advances in the determination the structure of T-ag have contributed much to our understanding of its role in catalyzing the initiation of DNA replication. We have focused our structural studies on the domain of T-ag that site specifically recognized the origin. Using x-ray crystallography, we determined that this domain forms a 'spiral-hexamer' and solved the co-structure of the T-ag-obd bound to an origin sub-fragment containing two pentanucleotides. These structures have significantly increased our understanding of the architectural arrangement of viral origins, the precise mechanism of origin recognition and provided critical insights into origin specific unwinding. However, additional structural information is now needed in order to understand the initiation process. Additional experiments led to the identification of the 'beta-hairpin1; a motif that is present in the initiators encoded by a broad spectrum of DNA tumor viruses. Preliminary studies indicated that this motif is needed of origin recognition, melting of duplex DNA and subsequent helicase activities. Therefore, we will establish the exact role(s) played by the "beta-hairpin" during initiation of viral replication. For example, we will extend recent studies indicating that the tip of the "beta-hairpin" plays an active role in melting origin DNA and that the ssDNA generated in this process is needed for subsequent assembly events. BRCT domains are known to recruit proteins involved in cell-cycle control, DNA repair and recombination. Our recent analyses indicate that a BRCT family member is present in the helicase domain of T-ag. Using techniques and approaches developed by individuals in the BRCT field, we will confirm that a BRCT motif is present in T-ag. This observation has the potential to greatly advance our understanding of how SV40 causes the myriad changes in the cells that it infects. Relevance to Public Health: Recent studies have demonstrated that the initiation of DNA replication in DNA tumor viruses is highly conserved. Therefore, a thorough description of initiation in one or two model systems will have broad ramifications in terms of our understanding of the propagation of diverse pathogens. These studies will also serve as a the paradigm when considering how DNA replication is initiated in higher-eukaryotic organisms. . Tufts University Boston, MA 02111 PHS 398 (Rev. 04/06) Page 2 ( Form Page 2 Principal Investigator/Program Director (Last, First, Middle): Bullock, Peter A. KEY PERSONNEL. See instructions. Use continuation pages as neededto provide the required information in the format shown below. Start with Principal Investigators). List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Bullock, Peter PBULLOCK1 Tufts University PI Bohm, Andrew andrewbohm Tufts University CO-PI Phelan, Paul n/a Tufts University Research Associate OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Human Embryonic Stem Cells ^ No Q Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://stemcells.nih.gov/reqistry/index.asp. Usecontinuation pages as needed. If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line PHS 398 (Rev. 04/06) Page 3 Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Bullock, Peter, A. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,